Systems and methods to detect rare mutations and copy number variation

Abstract

The present disclosure provides a system and method for the detection of rare mutations and copy number variations in cell free polynucleotides. Generally, the systems and methods comprise sample preparation, or the extraction and isolation of cell free polynucleotide sequences from a bodily fluid; subsequent sequencing of cell free polynucleotides by techniques known in the art; and application of bioinformatics tools to detect rare mutations and copy number variations as compared to a reference. The systems and methods also may contain a database or collection of different rare mutations or copy number variation profiles of different diseases, to be used as additional references in aiding detection of rare mutations, copy number variation profiling or general genetic profiling of a disease.

What is claimed is: 
     
       1. A method for detecting genetic aberrations in cell-free DNA (“cfDNA”) molecules from a subject, comprising:
 a) providing cfDNA molecules obtained from a bodily sample of the subject; 
 b) attaching tags comprising barcodes having a plurality of different barcode sequences to the cfDNA molecules to tag at least 20% of the cfDNA molecules, which attaching comprises ligating adaptors comprising the barcodes to both ends of the cfDNA molecules, wherein ligating comprises using more than 10× molar excess of the adaptors as compared to the cfDNA molecules, thereby generating tagged parent polynucleotides; 
 c) amplifying the tagged parent polynucleotides to produce amplified tagged progeny polynucleotides; 
 d) sequencing the amplified tagged progeny polynucleotides to produce a plurality of sequence reads from each of the tagged parent polynucleotides, wherein each sequence read of the plurality of sequence reads comprises a barcode sequence and a sequence derived from a cfDNA molecule of the cfDNA molecules; 
 e) mapping sequence reads of the plurality of sequence reads to one or more reference sequences from a human genome; 
 f) grouping the sequence reads mapped in e) into families based at least on barcode sequences of the sequence reads, each of the families comprising sequence reads comprising the same barcode sequence, whereby each of the families comprises sequence reads amplified from the same tagged parent polynucleotide; 
 g) at each of a plurality of genetic loci in the one or more reference sequences, collapsing sequence reads in each family to yield a base call for each family at the genetic locus; and 
 h) detecting, at one or more genetic loci, a plurality of genetic aberrations, wherein the plurality of genetic aberrations comprises two or more different members selected from the group of members consisting of a single base substitution, a copy number variation (CNV), an insertion or deletion (indel), and a gene fusion. 
 
     
     
       2. The method of  claim 1 , comprising providing less than 100 nanograms (ng) of the cfDNA molecules. 
     
     
       3. The method of  claim 1 , comprising providing less than 10 nanograms (ng) of the cfDNA molecules. 
     
     
       4. The method of  claim 1 , comprising providing between 100 and 100,000 human haploid genome equivalents of the cfDNA molecules, wherein the cfDNA molecules are tagged with between 2 and 1,000,000 unique identifiers. 
     
     
       5. The method of  claim 1 , comprising providing between 1,000 and 50,000 human haploid genome equivalents of the cfDNA molecules, wherein the cfDNA molecules are tagged with between 2 and 1,000 unique identifiers. 
     
     
       6. The method of  claim 1 , wherein each of the plurality of different barcode sequences is at least 5 nucleotides in length. 
     
     
       7. The method of  claim 1 , wherein the attaching comprises non-uniquely tagging the cfDNA molecules with at least 10 and at most 1,000 different barcode sequences. 
     
     
       8. The method of  claim 1 , wherein the attaching comprises uniquely tagging the cfDNA molecules. 
     
     
       9. The method of  claim 1 , wherein the attaching comprises performing blunt-end ligation or sticky end ligation. 
     
     
       10. The method of  claim 1 , wherein the attaching comprises non-uniquely tagging the cfDNA molecules such that no more than 5% of the tagged parent polynucleotides are uniquely tagged. 
     
     
       11. The method of  claim 1 , wherein at least 50% of the cfDNA molecules are tagged by the attaching. 
     
     
       12. The method of  claim 1 , wherein at least 80% of the cfDNA molecules are tagged by the attaching. 
     
     
       13. The method of  claim 1 , further comprising selectively enriching for polynucleotides mapping to one or more selected reference sequences prior to the sequencing, wherein the selectively enriching comprises (i) subjecting the cfDNA molecules to selective amplification against the one or more selected reference sequences, (ii) subjecting the tagged parent polynucleotides to selective amplification against the one or more selected reference sequences, (iii) subjecting the amplified progeny polynucleotides to selective sequence capture against the one or more selected reference sequences, or (iv) subjecting the cfDNA molecules to selective sequence capture against the one or more selected reference sequences. 
     
     
       14. The method of  claim 13 , wherein the selectively enriching comprises enriching for polynucleotides mapping to the following genes: V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), adenomatous polyposis coli (APC), and tumor protein 53 (TP53). 
     
     
       15. The method of  claim 1 , wherein sequencing comprises massively parallel sequencing. 
     
     
       16. The method of  claim 1 , wherein the amplified tagged progeny polynucleotides are sequenced to produce an average of 5 to 10 sequence reads for each family. 
     
     
       17. The method of  claim 1 , wherein the base call for each family possesses an error rate below 0.0001%. 
     
     
       18. The method of  claim 1 , wherein each base of the tagged parent polynucleotides has at least 99% chance of being represented by at least one sequence read among the sequence reads mapped in e). 
     
     
       19. The method of  claim 4 , wherein grouping the sequence reads mapped in e) is further based on one or more of: sequence information at a beginning of the sequence derived from the cfDNA molecule, sequence information at an end of the sequence derived from the cfDNA molecule, and length of the sequence read. 
     
     
       20. The method of  claim 4 , wherein grouping the sequence reads mapped in e) is further based on a plurality of: sequence information at a beginning of the sequence derived from the cfDNA molecule, sequence information at an end of the sequence derived from the cfDNA molecule, and length of the sequence read. 
     
     
       21. The method of  claim 1 , wherein at least one single base substitution is detected. 
     
     
       22. The method of  claim 1 , wherein the two or more members comprise a copy number variation (CNV). 
     
     
       23. The method of  claim 1 , wherein at least one indel is detected. 
     
     
       24. The method of  claim 1 , wherein at least one gene fusion is detected. 
     
     
       25. The method of  claim 1 , wherein at least one single base substitution and at least one copy number variation is detected. 
     
     
       26. The method of  claim 1 , further comprising detecting, at one or more genetic loci, one or more genetic aberrations selected from: a transversion, a translocation, an inversion, a deletion, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, chromosome fusions, a gene truncation, a gene amplification, a gene duplication, a chromosomal lesion, a DNA lesion, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns and abnormal changes in nucleic acid methylation. 
     
     
       27. The method of  claim 21 , wherein the single base substitution is detected with a sensitivity of at least 1%. 
     
     
       28. The method of  claim 21 , wherein the single base substitution is detected with a sensitivity of at least 0.1%. 
     
     
       29. The method of  claim 1 , wherein the plurality of genetic aberrations comprises three or more different members selected from the group of members consisting of a single base substitution, a copy number variation (CNV), an insertion or deletion (indel), and a gene fusion. 
     
     
       30. The method of  claim 1 , wherein the plurality of genetic aberrations comprises a single base substitution, a copy number variation (CNV), an insertion or deletion (indel), and a gene fusion. 
     
     
       31. The method of  claim 1 , wherein the plurality of genetic aberrations comprises a plurality of each of two or more different members selected from the group of members consisting of a single base substitution, a copy number variation (CNV), an insertion or deletion (indel), and a gene fusion. 
     
     
       32. The method of  claim 1 , wherein each of the tagged parent polynucleotides is uniquely tagged. 
     
     
       33. The method of  claim 1 , wherein each of the tagged parent polynucleotides is non-uniquely tagged.